scholarly journals Mutational analysis of a C-dependent late promoter of bacteriophage Mu.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 619-629 ◽  
Author(s):  
L W Chiang ◽  
M M Howe
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Bacteriophage Mu ◽  
C Protein ◽  
Compatible Plasmid ◽  
Late Transcription ◽  
Nested Sets ◽  
Rna Polymerase Promoter ◽  
Beta Galactosidase

Abstract Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.

scholarly journals DNA Unwinding Mechanism for the Transcriptional Activation ofmomP1Promoter by the Transactivator Protein C of Bacteriophage Mu

2001 ◽  
Vol 276 (50) ◽  
pp. 46941-46945 ◽  
Author(s):  
Shashwati Basak ◽  
Valakunja Nagaraja
Keyword(s):  
Conformational Changes ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Transcription Activation ◽  
Bacteriophage Mu ◽  
Promoter Elements ◽  
Spacer Length ◽  
C Protein ◽  
Transactivator Protein ◽  
Reduced Activity

Transcription factor-induced conformational changes in DNA are one of the mechanisms of transcription activation. C protein of bacteriophage Mu appears to transactivate themomgene of the phage by this mode. DNA binding by C to its site leads to torsional changes that seem to compensate for a weakmomP1 promoter having a suboptimal spacing of 19 bp between the poor −35 and −10 elements. The C-mediated unwinding could realign the promoter elements for RNA polymerase recruitment to the reoriented promoter. In this study, the model has been tested by mutational analysis of the spacer region ofmomP1 and by assessing the strength of the mutant promoters. The response to C-mediated transactivation was dependent on the spacer length of the promoters. Mutants with 17-bp spacing between the two promoter elements showed reduced activity in the presence of the transactivator as compared with their basal level. A synthetic promoter with near consensus promoter elements and optimal 17-bp spacing was also tested to evaluate the model. The results imply a role for C-mediated unwinding inmomtranscription activation.

scholarly journals MUTATIONAL ANALYSIS OF THE REGION SURROUNDING THE 93D HEAT SHOCK LOCUS OF DROSOPHILA MELANOGASTER

Genetics ◽  
1984 ◽  
Vol 106 (2) ◽  
pp. 249-265
Author(s):  
Jym Mohler ◽  
Mary Lou Pardue
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Γ Irradiation ◽  
Lethal Mutations ◽  
Complementation Groups ◽  
In Trans ◽  
Shock Locus ◽  
Shock Proteins

ABSTRACT The region containing subdivisions 93C, 93D and 93E on chromosome 3 of Drosophila melanogaster has been screened for visible and lethal mutations. Treatment with three mutagens, γ irradiation, ethyl methanesulfonate and diepoxybutane, has produced mutations that fall into 20 complementation groups, including the previously identified ebony locus. No point mutations affecting the heat shock locus in 93D were detected; however, a pair of deficiencies that overlap in the region of this locus was isolated. Flies heterozygous in trans for this pair of deficiencies are capable of producing all of the major heat shock puffs (except 93D) and the major heat shock proteins. In addition, these flies show recovery of normal protein synthesis following a heat shock.

scholarly journals Determinants That Control the Specific Interactions between TAB1 and p38α

2006 ◽  
Vol 26 (10) ◽  
pp. 3824-3834 ◽  
Author(s):  
Huamin Zhou ◽  
Min Zheng ◽  
Jianming Chen ◽  
Changchuan Xie ◽  
Anand R. Kolatkar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Transforming Growth Factor ◽  
Mutational Analysis ◽  
Specific Binding ◽  
Point Mutations ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
C Terminus

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

2006 ◽  
Vol 188 (4) ◽  
pp. 1411-1418 ◽  
Author(s):  
Guangnan Chen ◽  
Amrita Kumar ◽  
Travis H. Wyman ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Base Pair ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Dna Binding Protein ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Level Of Activity ◽  
High Level ◽  
Activity Dependent

ABSTRACT At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ΔabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of Pskf produced a promoter that was active constitutively in both ΔabrB and Δspo0A ΔabrB strains. Therefore, the base pair at position −9 of Pskf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

2000 ◽  
Vol 278 (3) ◽  
pp. L477-L484 ◽  
Author(s):  
Ramgopal Margana ◽  
Kiflu Berhane ◽  
M. Nurul Alam ◽  
Vijayakumar Boggaram
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Deletion Mapping ◽  
Clara Cells ◽  
Mobility Shift ◽  
Upstream Promoter ◽  
Upstream Promoter Region

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising −236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, −236 to −140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, −236 to −170 and −170 to −140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

scholarly journals Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

2021 ◽  
Vol 20 (3) ◽  
Author(s):  
Hairul Aini Hamzah ◽  
Rahmatullah Sirat ◽  
Mohammed Imad A. Mustafa Mahmud ◽  
Roesnita Baharudin
Keyword(s):  
Mutational Analysis ◽  
Single Point ◽  
Point Mutations ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Resistant Bacteria ◽  
Single Point Mutation ◽  
Phenotypic Resistance ◽  
Drug Effectiveness ◽  
E Coli

 Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

scholarly journals Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

1988 ◽  
Vol 8 (4) ◽  
pp. 1398-1407 ◽  
Author(s):  
M Guertin ◽  
H LaRue ◽  
D Bernier ◽  
O Wrange ◽  
M Chevrette ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transient Expression ◽  
Promoter Activity ◽  
Gene Activation ◽  
Point Mutations ◽  
Binding Assays ◽  
Recognition Sequence ◽  
Base Pairs ◽  
Receptor Recognition ◽  
Afp Promoter

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

scholarly journals Analysis of Promoters Recognized by HrpL, an Alternative σ-Factor Protein from Pantoea agglomerans pv. gypsophilae

2005 ◽  
Vol 18 (7) ◽  
pp. 634-643 ◽  
Author(s):  
Gal Nissan ◽  
Shulamit Manulis ◽  
Dan M. Weinthal ◽  
Guido Sessa ◽  
Isaac Barash
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Present Report ◽  
Pantoea Agglomerans ◽  
Computational Techniques ◽  
Promoter Strength ◽  
Significant Activity ◽  
Gall Formation ◽  
Consensus Motif ◽  
Σ Factor

HrpL, an alternative σ factor, activates the transcription of the Hrp regulon by its binding to a common “hrp box” promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5′-GGAACC-N15–16-CCACNNA-3′. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the -35 consensus motif (GGAAC) and the 3 nt of the -10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the -35 consensus motif eliminated HrpL binding, whereas mutations in the -10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P. agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

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